1. Field of the Invention
The present invention relates generally to the fields of cardiovascular disease and hypertension. More particularly, it concerns antisense oligonucleotide compositions and methods that are useful for reducing hypertension, cardiac hypertrophy, and myocardial ischemia in animals, particularly in mammals such as humans. Specifically, the invention provides antisense oligonucleotide and polynucleotide compositions capable of binding to xcex21-adrenoceptor (xcex21-adrenergic receptor, xcex21-AR)-specific mRNA and inhibiting translation of the xcex21-adrenoceptor mRNA, thereby decreasing the number of xcex21-AR polypeptides in cells capable of expressing this mRNA. Disclosed are antisense oligonucleotide and peptide nucleic acid compositions, pharmaceutical formulations thereof, and vectors encoding antisense oligonucleotides that specifically bind xcex21-AR mRNA and alter expression of the mRNA in a host cell.
2. Description of Related Art
1.2.1 Hypertension
Hypertension is the result of increased arterial resistance to blood flow and left untreated can lead to various pathological consequences. Hypertension affects approximately 40 million people in the United States. Heart attack (Nicholls et al., 1998), kidney damage (Agodoa, 1998), stroke (Chamorro et al., 1998) and loss of vision (Satllworth and Waldron, 1997) are typical conditions that result from high blood pressure. When blood vessels are subjected to high pressure for extended periods of time, they respond by thickening, vasospasm, and internal build-up of lipids and plaques, a condition known as arteriosclerosis. Arteriosclerosis further causes a decreased blood flow to the kidneys, which respond by releasing the protease renin. An overactive renin-angiotensin system is often implicated in the development of hypertension and cardiovascular disease (Nicholls et al., 1998).
Hypertension is often called the xe2x80x9csilent killer,xe2x80x9d since half of the population afflicted with high blood pressure are unaware of the condition. Thus, an initial step in combating hypertension is early detection. Following diagnosis, actions must be taken to control the disorder.
1.2.2 Treatment of Hypertension
Currently, four major categories of hypertensive drugs are administered to treat high blood pressure: (1) Diuretics, typically the drug of choice when the abnormal blood pressure is not very high, increase the rate at which the body eliminates urine and salt, resulting in decreased blood pressure by reducing volume (Moser, 1998); (2) xcex2-adrenergic blockers (xcex2-blockers), typically prescribed in combination with diuretics, lower blood pressure and heart rate (Rodgers, 1998); (3) Calcium channel blockers work by preventing the entry of calcium into cells, which reduces vasoconstriction (Rosenthal, 1993); (4) angiotensin converting enzyme (ACE) inhibitors prevent the narrowing and constriction of blood vessels by blocking the production of the vasoconstrictive peptide angiotensin II (AT-II), a product of ACE (Rosenthal, 1993).
Unfortunately, the short lasting effects of these drugs often require multiple, even daily doses to be therapeutically effective. Poor compliance is a major problem with drug regimens and can lead to a hypertensive crisis if the drug is not taken as scheduled.
The sympathetic nervous system plays a crucial role in the regulation of blood pressure (BP), mainly through activating xcex1- and xcex2-adrenergic receptors xcex21-ARs) in the effector organs, including heart, kidney, and blood vessels. Adrenergic-blocking agents, especially xcex2-blockers, are commonly used in the treatment of hypertension, ischemic heart disease, and arrhythmia (reviewed in Sproat and Lopez, 1991). However, xcex2-blockers cause several side effects, which are usually associated with their central nervous system (CNS) reaction (e.g., sleep disturbance, depression, impotence, dizziness and fatigue) and xcex22-adrenergic antagonistic activity (e.g., increase in peripheral vascular resistance, worsening of asthma symptoms). In addition, because of their short half-life (3 to 10 hrs), xcex2-blockers must be taken daily to be effective. Because a cardiovascular disease such as hypertension is a life-lone disorder, longer-lasting treatment without side effects would be desirable.
Although the precise mechanism underlying the antihypertensive effects of xcex2-blockers remains unclear, it is generally accepted that they antagonize the xcex21-AR activity in heart and kidney, decreasing cardiac output and plasma renin activity (Sproat and Lopez, 1991). A new approach to xcex21-blockade has been designed that reduces the number of receptors. Antisense oligonucleotides (AS-ODN) or antisense DNA designed specifically against B1-ARs might represent a new class of xcex2-blockers. Antisense techniques, through a number of mechanisms (Phillips et al., 1997), can effectively downregulate the expression of target proteins. Clinical trials using antisense in targeting AIDS (Akhtar and Rossi, 1996), cancer (Dachs et al., 1997), and other genetic and acquired diseases (Yla, 1997) indicate their potential clinical usefulness. The antisense approach has several potential advantages over xcex2-blockers. First, the specificity of AS-ODNs is based on DNA sequence. Second, AS-ODNs do not have direct CNS effects, because of the negligible transport of these highly polar molecules through the blood-brain barrier (Agrawal et al., 1991). Third, antisense elements tested in different systems produce long-term effects after single treatment (Gyurko et al., 1997). This prolonged effect can be attributed to 2 features of AS-ODN. One is the extended half-life of chemically modified ODN. The half-life 15-20-mer phosphorothioated ODN is 20 to 50 hrs in rats and mice after intravenous injection (Iversen, 1991; Zhang et al., 1995). The other is associated with the nature of antisense inhibition, which provides a delayed yet prolonged blockade of target proteins distinct from the direct competitive antagonists currently available.
1.2.3 xcex21-Adrenoceptors
xcex21-ARs, which are distributed in the heart, kidney and blood vessels, play a role in the physiological control of blood pressure. For many years xcex2-blocker drugs have been used for the treatment of hypertension through a regimen of daily dosing. The mechanism of control of blood pressure is not precisely known but the value of beta-blockers in hypertension control has been underscored by the reports of the Joint National Committee on High Blood Pressure recommending xcex2-blockers as the first line of defense in the treatment of hypertension. Current xcex2-blocker drugs, however, have certain disadvantages, including: (1) they have to be taken daily, or twice a day and compliance is a problem; (2) they have central effects, leading to psychological changes that contribute to the problem of patient compliance; and (3) many of the xcex2-blockers now available are not specific for xcex21-ARs and, therefore, can have untoward side effects.
As can be understood from the above, there remains a need for an effective xcex21-AR blocker that is highly specific, nontoxic, produces few side effects and does not cross the blood brain barrier to produce psychological changes. Also, a xcex2-blocker that would last several days or weeks would allow patients more flexibility in the regimen of drug dosage by taking drugs infrequently. Thus, the need exists for an effective treatment of cardiac deficiencies (including hypertension, hypertrophy, ischemias, and the like) that circumvents the toxic side effects of existing therapies and provides more specific xcex21-AR inhibition with longer acting effects for improved patient compliance. In addition, methods for delivery of antisense oligonucleotides and polynucleotides to a host cell, and in particular, non-invasive administration of specific antisense constructs to a mammal such as a human subject is particularly desirable.
The present invention overcomes these and other limitations in the prior art by providing antisense nucleic acid compounds and compositions comprising them that specifically inhibit or reduce the expression of an mRNA encoding a xcex21-adrenoceptor (xcex21-AR) polypeptide in a host cell expressing the mRNA. More specifically, the subject invention provides antisense oligonucleotides (AS-ONs) and antisense oligo-peptide nucleic acids (AS-PNAs) that can specifically bind to a mammalian xcex21-AR mRNA, resulting in the reduction or inhibition of translation of the messenger ribonucleic acid (mRNA) into xcex21-AR polypeptide. Such oligonucleotides and PNA may be readily formulated in pharmaceutically-acceptable vehicles and provided directly to host cells, or administered systemically to mammals that express xcex21-AR mRNA to reduce the level of xcex21-AR produced in such cells and affected mammals.
The invention also provides genetic constructs comprising substantially full-length, antisense polynucleotide (AS-PN) sequences operably linked to a suitable promoter that may be used to transform selected cells to endogenously express xe2x80x9cantisensexe2x80x9d mRNA sequences that are complementary to the native xcex21-AR mRNA sequence. When expressed in a suitable host cell, these essentially full length anti-mRNAs specifically bind to the native xcex21-AR mRNA produced in the same host cell, and effectively reduce the availability of native xcex21-AR mRNA that can serve as a template for the translation machinery of the host cell to produce xcex21-AR polypeptide.
The antisense compounds and the genetic constructs of the present invention may be prepared in a variety of compositions, and may also be formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects to inhibit or significantly reduce the expression of xcex21-AR-specific mRNA, and/or to inhibit or significantly reduce the translation of xcex21-AR-specific mRNA into functional xcex21-AR polypeptide. The antisense compounds of the present invention, and compositions comprising them provide new and useful therapeutics for the treatment, control, and amelioration of symptoms of a variety of cardiovascular disorders including hypertension, ischemia, cardiac hypertrophy, myocardial infarction, cardiac dysfunction, and diseases of the heart, that result from, or are exacerbated by, expression of xcex21-AR-specific mRNA in the host cells of the affected mammal. Moreover, pharmaceutical compositions comprising one or more of the nucleic acid compounds disclosed herein, provide significant advantages over existing conventional therapiesxe2x80x94namely, (1) their reduced side effects, (2) their increased efficacy for prolonged periods of time, (3) their ability to increase patient compliance due to their ability to provide therapeutic effects following as little as a single dose of the composition to affected individuals.
Preferred antisense compounds for use in the practice of the present invention are those nucleic acid and peptide nucleic acid sequences that specifically bind to a gene or an mRNA encoding xcex21-AR polypeptide and that inhibit the expression or reduce the level of xcex21-AR polypeptide in a mammalian host cell that expresses the gene and/or the mRNA.
The compounds of the invention, and the pharmaceutical formulations thereof, permit the development of methods for treating hypertension, ischemia, cardiac hypertrophy, cardiac dysfunction, or other cardiovascular diseases through the administration of at least one antisense compound that specifically binds to a mammalian xcex21-AR-specific gene or mRNA, wherein the binding of the antisense compound to the gene or the mRNA alters, decreases, inhibits, and/or prevents transcription of the xcex21-AR-specific mRNA, or, alternatively alters, decreases, inhibits, and/or prevents translation of the xcex21-AR-specific mRNA into functional xcex21-AR polypeptide in a host cell. As described above, the present methods offer significant advantages over traditional pharmacological modalities involving drugs that block or interfere with activity of the xcex21-AR polypeptide (and not the amount of xcex21-AR polypeptide). The present methods also avoid many of the untoward side effects of conventional therapies, avoid invasive surgical procedures, and are effective in lower, less frequent dosing regimens. The ability to dose less frequently represents a key aspect of improving patient compliance in dosing and reduces administration costs associated with more frequent dosage regimens.
In a first embodiment, the invention provides antisense oligonucleotides that specifically bind to a mammalian xcex21-AR-specific gene or a mammalian xcex21-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, xcex21-AR polypeptide produced in the cell.
In a second embodiment, the invention provides antisense peptide nucleic acids that specifically bind to a mammalian xcex21-AR-specific gene or a mammalian xcex21-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, xcex21-AR polypeptide produced in the cell.
In a third embodiment, the invention provides antisense polynucleotides that specifically bind to a mammalian xcex21-AR-specific gene or to a mammalian xcex21-AR-specific mRNA in a host cell, and alter the expression of, or quantity of, xcex21-AR polypeptide produced in the cell. The antisense polynucleotides represent full-length, or substantially full-length sequences that are complementary to a mammalian xcex21-AR-specific mRNA, and that when present in a cell that expresses a mammalian xcex21-AR-specific mRNA, will specifically bind to the xcex21-AR-specific mRNA, thereby reducing the availability of functional mRNA in the cell from which the cellular protein machinery can translate functional xcex21-AR polypeptide. Preferably, these full-length, or substantially full-length polynucleotides are provided to a cell via a genetic construct that comprises a promoter capable of expressing the complementary mRNA in a selected host cell. Such genetic constructs may be provided to suitable host cells using any one of the conventional gene therapy modalities known to those of skill in the art, such as, for example by one or more of the viral, retroviral, adenoviral, or adenoassociated viral constructs commonly exploited for the delivery and expression of heterologous polynucleotides.
In a fourth embodiment, the invention provides compositions that comprise one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds and a suitable diluent, carrier, or pharmaceutical vehicle.
In a fifth embodiment, the invention provides pharmaceutical formulations that comprise one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds and at least one antihypertensive pharmaceutical compound.
In a sixth embodiment, the invention provides pharmaceutical compositions that comprise at least two or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds.
In a seventh embodiment, the invention provides pharmaceutical compositions that comprise at least two or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compounds in combination with at least one antihypertensive pharmaceutical compound.
In an eighth embodiment, the invention provides therapeutic kits and medicaments that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the treatment of mammalian cardiac dysfunction or disease. Alternatively, the invention provides kits that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the preparation of genetic constructs for the development of suitable gene therapy vectors. Likewise, the invention provides kits that comprise at least one or more of the disclosed antisense oligonucleotides, polynucleotides, and peptide nucleic acid compositions in combination with instructions for using the compositions in the formulation of multi-drug xe2x80x9ccocktailsxe2x80x9d for the treatment of one or more pathological conditions.
The invention also provides methods for the treatment of one or more cardiac diseases or dysfunctions employing one or more of the antisense compounds or compositions as described herein.
In each of the embodiments described herein, the oligonucleotide, polynucleotide, or peptide nucleic acid compound comprises a sequence region least 9 to about 35 bases in length, wherein the oligonucleotide specifically binds to a portion of mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide, and further wherein binding of the oligonucleotide to the mRNA is effective in decreasing the activity of xcex21-AR in a host cell expressing the mRNA.
In oligonucleotide and oligo-peptide nucleic acid embodiments, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least nine, at least ten, at least eleven, at least twelve, at least thirteen at least fourteen, or at least fifteen contiguous bases from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, as well as the smaller n-mer sequences illustrated in SEQ ID NO:29 through SEQ ID NO:186.
Alternatively, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four, or at least twenty-five contiguous nucleotides from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.
Alternatively, the oligonucleotide or PNA preferably consists of a sequence of from about nine to about 35 nucleotides in length, and preferably comprises a sequence of at least twenty-six, at least twenty-seven, at least twenty-eight, at least twenty-nine, at least thirty, at least thirty-one, at least thirty-two, at least thirty-three, or at least thirty-four contiguous nucleotides from any one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.
Alternatively, the oligonucleotide or PNA may consist of a sequence of from about nine to about 35 nucleotides in length, and may comprise the entire sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.
The oligonucleotide or PNA compound may consist essentially of the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186.
The oligonucleotide or PNA compound may also consist of the sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186.
As described above, the length of the preferred oligonucleotide or PNA compounds preferably will be on the order of from about nine to about 35 or so nucleotides. As such, in addition to those compounds that are nine nucleotides in length, and those compounds that are 35 nucleotides in length, all intermediate size compounds are also contemplated to be useful in the practice of the present invention. Thus, oligonucleotide or PNA compounds that are ten nucleotides in length, eleven nucleotides in length, twelve nucleotides in length, thirteen nucleotides in length, fourteen nucleotides in length, fifteen nucleotides in length, sixteen nucleotides in length, seventeen nucleotides in length, eighteen nucleotides in length, nineteen nucleotides in length, twenty nucleotides in length, twenty-one nucleotides in length, twenty-two nucleotides in length, twenty-three nucleotides in length, twenty-four nucleotides in length, twenty-five nucleotides in length, twenty-six nucleotides in length, twenty-seven nucleotides in length, twenty eight nucleotides in length, twenty-nine nucleotides in length, thirty nucleotides in length, thirty-one nucleotides in length, thirty-two nucleotides in length, thirty-three nucleotides in length, and thirty-four nucleotides in length are also contemplated to fall within the scope of the present disclosure. In fact, the preferred oligonucleotide or PNA compounds may also be slightly shorter than the preferred range, that is, they may be about six or about seven or about eight nucleotides in length, or they may even be slightly longer than the preferred range (i.e., they may be about thirty-six or about thirty-seven or even about thirty-eight or so nucleotides in length), and may still function to reduce the level of xcex21-AR polypeptide in a cell, and thus, effective in the treatment of cardiac disorders resulting from an elevated level of xcex21-AR polypeptide.
Likewise, there is no obligate requirement that the antisense compounds be exactly 100% complementary to a given target sequence on the mammalian xcex21-AR mRNA. Nor is there an obligate requirement that the antisense compounds be exactly 100% identical to one of the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186. In fact, the only requirement is that the oligonucleotide or PNA compound have sufficient homology to the gene or the mRNA encoding xcex21-AR polypeptide so that upon specifically binding to the complementary region, a reduction in either the transcription of the xcex21-AR gene and/or a reduction in the translation of xcex21-AR polypeptide from the mRNA is observed. Therefore, the compounds may have 4 or fewer, 3 or fewer, 2 or fewer, or even 1 mismatch from the target sequence to which it specifically binds, and may still be sufficiently active to cause a reduction in xcex21-AR polypeptide within the host cell. Thus, in addition to those compounds disclosed above that comprise sequences that are 100% identical to the sequences disclosed in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53, or any one or more of the smaller n-mer sequences illustrated in SEQ ID NO:54 through SEQ ID NO:186, the invention also encompasses antisense compounds that comprise sequence regions that are about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, or even about 90% identical to a portion of one of those sequences, so long as the resulting degenerate nucleotide sequence retains sufficient homology so that it specifically binds to the gene or mRNA encoding xcex21-AR polypeptide.
In certain embodiments, the polynucleotide comprises at least 9 contiguous bases from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53. In other aspects of the invention, the polynucleotide comprises DNA, RNA, PNA, or preferably a derivatized or modified polynucleotide, including phosphorothioate derivates and the like.
Preferably the antisense polynucleotide compound comprises a sequence that is substantially the full-length complement of the xcex21-AR mRNA, wherein binding of the substantially full-length complementary sequence to the xcex21-AR mRNA is effective in reducing the translation of the mRNA into the xcex21-AR polypeptide. Preferably the substantially full-length complementary sequence is comprised within a suitable vector that may be delivered to appropriate host cells. The vector preferably comprises a nucleic acid segment that is operably linked to a promoter sequence that expresses the complementary antisense sequence in the host cell. This complementary near full-length sequence is then able to specifically bind to the substantially full length xcex21-AR mRNA and thereby prevent or substantially reduce the translation of the mRNA into xcex21-AR polypeptide. The term substantially full-length is meant to include those sequences that comprise a sequence that is complementary to a region that is at least about 80% or more of the native xcex21-AR-specific mRNA. Thus, for a xcex21-AR mRNA that is 1000 nucleotides in length, a suitable complementary substantially full-length complement is a sequence that is complementary to a region of at least 800 nucleotides of the particular xcex21-AR mRNA. Likewise, all sequences that are greater than 80% of the full-length sequences (i.e. those that are at least 81% full length, at least 82% full length, at least 83% full length, at least 84% full length, at least lo 85% full length, at least 86% full length, at least 87% full length, at least 88% full length, at least 89% full length, at least 90% full length, at least 91% full length, at least 92% full length, at least 93% full length, at least 94% full length, at least 95% full length, at least 96% full length, at least 97% full length, at least 98% full length, and at least 99% full length are also contemplated to fall within the scope of the present disclosure.
When desirable, the clinician may combine two or more of the oligonucleotide, polynucleotide, or PNA compounds disclosed above to provide an antisense xe2x80x9ccocktailxe2x80x9d to effect a more substantial reduction in the levels of xcex21-AR polypeptide. Likewise, the patient or animal to be treated may benefit from a combination therapy involving one or more of the oligonucleotide, polynucleotide, or PNA compounds disclosed herein and at least one anti-hypertensive or other cardiac therapy medicament.
For example, the composition may compris at least a first anti-hypertensive agent. In particular embodiments, the anti-hypertensive agent is selected from the group consisting of captopril (Captopril(copyright)), enalapril (Vasotec(copyright)), ramipril (Altace(copyright)), fosinopril (Monopril(copyright)), lisinopril (Prinivil(copyright), Zestril(copyright)), quinapril (Accupril(copyright)), benazepril (Lotensin(copyright)), trandolapril (Mavik(copyright)), and moexipril (Univasc(copyright)). In other embodiments, the anti-hypertensive agent is selected from the group of angiotensin receptor blockers consisting of candesartan (Atacand(copyright)), losartan (Cozaar(copyright) and Hizaar(copyright)), valsartan (Diovan(copyright)), and irbesartan (Avapro(copyright)).
In still other embodiments, the anti-hypertensive agent is selected from the group of diuretic consisting of dichlorphenamide (Daranide(copyright)), spironolactone (Aldactazide(copyright)), hydrochlorothiazide (Microzide(copyright) and Dyazide(copyright)), triamterene (Maxzide(copyright)), amiloride (Midamor(copyright) and Moduretic(copyright)), torsemide (Demadex(copyright)), ethacrynice acid (Edecrin(copyright)), furosemide (Lasix(copyright)), hydroflumethiazide (Diucardin(copyright)), chlorothiazide (Diuril(copyright)), methylclothiazide (Enduron(copyright)), polythiazide (Renese(copyright)), chlorthalidone (Thalitone(copyright)) and metolazone (Zaroxolyn(copyright)).
In still further embodiments, the anti-hypertensive agent is selected from the group of calcium channel blockers consisting of nifedepine (Adalat(copyright) and Procardia(copyright)), verapamil (Isoptin(copyright), Verelan(copyright), Calan(copyright) and Covera(copyright)), nicardipine (Cardene(copyright)), diltiazem (Tiazac(copyright), Cardizem(copyright) and Dilacor(copyright)), isradipine (DynaCirc(copyright)), nimodipine (Nimotop(copyright)), amlodipine (Norvase(copyright)), felodipine (Plendil(copyright)), misoldipine (Sular(copyright)), and bepridil (Vasocor(copyright)).
In certain preferred embodiments, the composition may further comprise one or more pharmaceutically acceptable vehicles, exemplified by, but not limited to, liposomes, lipid particles, lipid vesicles, nanoparticles, microparticles, nanocapsules, nanospheres, and sphingosomes to facilitate administration of the antisense composition(s) to the affected patient or animal.
In certain aspects of the invention, the antisense composition of the present invention is specific for an mRNA encoding the human xcex21-AR polypeptide. In particular embodiments, the host cell is a mammalian host cell. In certain preferred embodiments of the invention, the host cell is a human cell. In other preferred aspects, the host cell is comprised within a human.
The present invention also provides compositions that comprise at least a first antisense oligonucleotide, PNA, or polynucleotide specific for a mammalian xcex21-AR-specific mRNA as described above, and at least a second antisense compound specific for a mammalian renin, angiotensin, angiotensinogen, angiotensin type 1 (AT-1) receptor mRNA, or an ACE polypeptide.
In certain aspects of the invention, the second antisense compound is specific for a mammalian angiotensinogen mRNA, or an mRNA that encodes one or more RAS pathway-specific enzymes, such as a mammalian ACE polypeptide. In other aspects, the second antisense compound is specific for an mRNA that encodes a transcriptional factor.
Therapeutic combinations of three or more antisense compound are also provided. These cocktail therapies may comprise at least two antisense compounds specific for a mammalian xcex21-AR-encoding gene or mRNA, and at least a third antisense compound specific for the same mRNA, or alternatively, an mRNA encoding another polypeptide in the RAS pathway as described above. The third antisense compound may be specific for renin, AT-1 receptor, ACE, or angiotensinogen, or another gene or mRNA involved in biochemical pathways involved in producing or regulating blood pressure and/or causing or contributing to hypertension, ischemia, cardiac hypertrophy, or other cardiac dysfunction in an affected animal. Alternatively, the constructs may even be specific for one or more particular transcription factor(s) that regulate one or more genes involved in producing hypertension, ischemia, cardiac hypertrophy, or other cardiac dysfunction in a mammal. Such combined therapy approached using two or more antisense oligonucleotides are particularly desirable where enhanced or synergistic activity towards treating hypertension ischemia, cardiac hypertrophy, or other cardiac dysfunction is achieved.
The invention further provides a method for reducing expression of a gene encoding manmmalian xcex21-AR polypeptide in a host cell, the method comprising providing to the host cell an amount of an antisense oligonucleotide, polynucleotide, or peptide nucleic acid that specifically binds to an mRNA encoding the xcex21-AR polypeptide, effective to reduce the amount of xcex21-AR polypeptide in the cell.
Additionally, the invention provides a method for reducing the number of mammalian xcex21-AR polypeptides in a cell, the method comprising introducing into the cell at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid that specifically binds to all, substantially-all, or a portion of the mRNA expressed from a gene encoding a mammalian xcex21-adrenoceptor polypeptide, and further wherein binding of the first antisense oligonucleotide, polynucleotide, or peptide nucleic acid to the mRNA is effective in reducing the number of mammalian xcex21-AR polypeptides in the host cell expressing the mRNA.
The invention also provides a method for decreasing or treating hypertension in an animal, the method comprising administering to the animal an effective amount of at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid, wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide, and further wherein binding of the compound to the mRNA is effective in decreasing the number of such polypeptides in a host cell expressing the mRNA.
The invention additionally provides a method for treating a disease associated with elevated xcex21-AR activity in a mammal, the method comprising administering to the animal an effective amount of at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid compound wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide, and further wherein binding of the compound to the mRNA is effective in decreasing the receptor activity or receptor number in a host cell expressing the mRNA, such that a decrease in xcex21-adrenoceptor activity is effected, thereby resulting in amelioration of the disease that results from, or is exacerbated by, an elevated level of xcex21-AR activity in the affected animal.
The invention also provides a method for treating a cardiac hypertrophic or ischemic disease that is associated with, that results from, or is exacerbated by, the presence of, or an elevation in, xcex21-adrenoceptor polypeptides in the affected animal. This method generally involves the administration to the animal of one or more compositions that comprise at least a first antisense oligonucleotide, polynucleotide, or peptide nucleic acid compound, wherein the compound specifically binds to all, substantially all, or a portion of the mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide in an amount, and for a time sufficient to decrease the number, amount, or activity of the xcex21-adrenoceptor polypeptide in a host cell expressing the mRNA.
Further provides are kits for treating hypertension in a human comprising: (a) a pharmaceutically-acceptable formulation comprising at least a first oligonucleotide of at least 9 to about 35 bases in length, wherein the oligonucleotide specifically binds to a portion of mRNA expressed from a gene encoding a mammalian xcex21-AR polypeptide, and further wherein binding of the oligonucleotide to the mRNA is effective in decreasing the activity, amount, or number of receptor polypeptides in a host cell expressing the mRNA; a pharmaceutical excipient; and (b) instructions for using the kit.
In addition to methods involving the delivery of exogenous oligonucleotide compositions to a host cell, or administration of such compositions to an animal in a therapeutic pharmaceutical formulation, the present invention also concerns gene therapy methods for introducing into a host cell a DNA construct that is transcribed by the cell machinery to give rise to an antisense RNA molecule that specifically binds to a portion of an mRNA encoding a mammalian xcex21-AR polypeptide. In a preferred embodiment, such therapies may be accomplished through the use of viral vectors, such as retro-, adeno- or adeno-associated viruses as described hereinbelow.
Regulation of expression of a gene encoding a mammalian xcex21-AR polypeptide in a mammalian cell genomes may also be achieved by integration of a gene under the transcription control of a promoter which is functional in the host and in which the transcribed strand of DNA is complementary to the strand of DNA that is transcribed from the endogenous xcex21-AR-specific polynucleotide sequence(s) one wishes to regulate. The integrated gene, referred to as an antisense gene, provides an RNA sequence capable of binding to naturally existing RNAs, exemplified by mammalian xcex21-AR polypeptide-specific mRNA, and inhibiting its expression, where the anti-sense sequence may bind to the coding, non-coding, or both, portions of the RNA. The antisense construction may be introduced into the animal cell in a variety of ways and be integrated into the animal genome for inducible or constitutive transcription of the antisense sequence.
Another aspect of the invention provides a pharmaceutical composition useful for inhibiting expression of mammalian xcex21-AR-specific mRNA comprising a pharmaceutical carrier and one or more antisense oligonucleotides of the present invention that specifically bind to and reduce expression of the specific mRNA. Another aspect of the invention provides a method for treating hypertension in a human comprising administering to a subject an effective amount of at least one oligonucleotide composition as described herein, in an amount effective to reduce hypertension in the human, or to ameliorate the degree or extent of hypertension in the patient.
The term xe2x80x9cxcex21-ARxe2x80x9d refers to polypeptides having amino acid sequences which are substantially similar to the native mammalian xcex21-AR polypeptide amino acid sequences and which are biologically active and/or which cross-react with xcex21-AR polypeptide-specific antibodies raised against a mammalian xcex21-AR polypeptide or peptide fragment thereof.
The term xe2x80x9cxcex21-ARxe2x80x9d also includes analogs of mammalian xcex21-AR polypeptides that exhibit at least some biological activity in common with native mammalian xcex21-AR polypeptides.
Furthermore, those skilled in the art of mutagenesis will appreciate that other analogs, as yet undisclosed or undiscovered, may be used to construct mammalian xcex21-AR polypeptide analogs or xcex21-AR fusion proteins or identify xcex21-AR-related mRNAs and/or genes using well-known molecular biology techniques, including those described herein. Oligonucleotides complementary to mammalian xcex21-AR polypeptide-encoding mRNAs form the heart of the present invention, especially human xcex21-AR polypeptide-encoding mRNAs.
The oligonucleotides (or xe2x80x9cODNsxe2x80x9d or xe2x80x9cpolynucleotidesxe2x80x9d or xe2x80x9coligosxe2x80x9d or xe2x80x9coligomersxe2x80x9d or xe2x80x9cn-mersxe2x80x9d) of the present invention are preferably deoxyoligonucleotides (ie. DNAs), or derivatives thereof; ribo-oligonucleotides (i.e. RNAs) or derivatives thereof; or peptide nucleic acids (PNAs) or derivatives thereof.
The term xe2x80x9csubstantially complementary,xe2x80x9d when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an Mrna encoding a mammalian xcex21-AR polypeptide. As such, typically the sequences will be highly complementary to the Mrna xe2x80x9ctargetxe2x80x9d sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the complementary portion of the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e. be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region of the Mrna and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target Mrna sequence into polypeptide product.
Substantially complementary oligonucleotide sequences will be greater than about 80 percent complementary (or xe2x80x98% exact-matchxe2x80x99) to the corresponding Nrna target sequence to which the oligonucleotide specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary oligonucleotide sequences for use in the practice of the invention, and in such instances, the oligonucleotide sequences will be greater than about 90 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding Mrna target sequence to which the oligonucleotide specifically binds, and even up to and including 96%, 97%, 98%, 99%, and even 100% exact match complementary to all or a portion of the target Mrna to which the designed oligonucleotide specifically binds.
Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
Table 1 lists representative oligonucleotide sequences contemplated for use in the practice of the present invention.
N means any nucleotide, e.g., C, A, U (T), or G.
Sequences in bold correspond to portions of the xcex21-AR open reading frame.
In addition to the indicated 35-mers, smaller internal n-mers that comprise from at least 9 bases in length up to the full length 35-mers listed are also contemplated to be useful in the practice of the present invention. For example, in addition to the first indicated full-length oligomer, all internal n-mers are also considered to fall within the scope of this disclosure. Thus for each of the 35-mers (SEQ ID NO:4 to SEQ ID NO:28) all internal 34-mers of each sequence as well as all internal 33-mers, 32-mers, 31-mers, 30-mers, 29-mers, 28-mers, 27-mers, 26-mers, 25-mers, 24-mers, 23-mers, 22-mers, 21-mers, 20-mers, 19-mers, 18-mers, 17-mers, 16-mers, 15-mers, 14-mers, 13-mers, 12-mers, 11-mers, 10-mers, and 9-mers, that are comprised within of each of the disclosed 35-mers are also contemplated to be useful in the practice of the present invention.
For illustrative purposes, all representative n-mers of SEQ ID NO:10 would include the following internal contiguous 9-mer to 34-mer sequences:
CAGCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:29)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCCTG (SEQ ID NO:30)
AGCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:31)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCCT (SEQ ID NO:32)
GCTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:33)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGTCC (SEQ ID NO:34)
CTCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:35)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGTC (SEQ ID NO:36)
TCGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:37)
GCAGCTCGGCATGGGCGCGGGGGTGCTCGT (SEQ ID NO:38)
CGGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:39)
GCAGCTCGGCATGGGCGCGGGGGTGCTCG (SEQ ID NO:40)
GGCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:41)
GCAGCTCGGCATGGGCGCGGGGGTGCTC (SEQ ID NO:42)
GCATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:43)
GCAGCTCGGCATGGGCGCGGGGGTGCT (SEQ ID NO:44)
CATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:45)
GCAGCTCGGCATGGGCGCGGGGGTGC (SEQ ID NO:46)
ATGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:47)
GCAGCTCGGCATGGGCGCGGGGGTG (SEQ ID NO:48)
TGGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:49)
GCAGCTCGGCATGGGCGCGGGGGT (SEQ ID NO:50)
GGGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:51)
GCAGCTCGGCATGGGCGCGGGGG (SEQ ID NO:52)
GGCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:53)
GCAGCTCGGCATGGGCGCGGGG (SEQ ID NO:54)
GCGCGGGGGTGCTCGTCCTGG (SEQ ID NO:55)
GCAGCTCGGCATGGGCGCGGG (SEQ ID NO:56)
CGCGGGGGTGCTCGTCCTGG (SEQ ID NO:57)
GCAGCTCGGCATGGGCGCGG (SEQ ID NO:58)
GCGGGGGTGCTCGTCCTGG (SEQ ID NO:59)
GCAGCTCGGCATGGGCGCG (SEQ ID NO:60)
CGGGGGTGCTCGTCCTGG (SEQ ID NO:61)
GCAGCTCGGCATGGGCGC (SEQ ID NO:62)
GGGGGTGCTCGTCCTGG (SEQ ID NO:63)
GCAGCTCGGCATGGGCG (SEQ ID NO:64)
GGGGTGCTCGTCCTGG (SEQ ID NO:65)
GCAGCTCGGCATGGGC (SEQ ID NO:66)
GGGTGCTCGTCCTGG (SEQ ID NO:67)
GCAGCTCGGCATGGG (SEQ ID NO:68)
GGTGCTCGTCCTGG (SEQ ID NO:69)
GCAGCTCGGCATGG (SEQ ID NO:70)
GGTGCTCGTCCTGG (SEQ ID NO:71)
GCAGCTCGGCATG (SEQ ID NO:72)
GTGCTCGTCCTGG (SEQ ID NO:73
GCAGCTCGGCAT (SEQ ID NO:74)
TGCTCGTCCTGG (SEQ ID NO:75)
GCAGCTCGGCA (SEQ ID NO:76)
GCTCGTCCTGG (SEQ ID NO:77)
GCAGCTCGGC (SEQ ID NO:78)
CTCGTCCTGG (SEQ ID NO:79)
GCAGCTCGG (SEQ ID NO:80), Etc.
In similar fashion, for illustrative purposes, and in the sake of brevity, representative n-mers of SEQ ID NO:1 would include the following internal contiguous sequences:
CGCGCCCATGCCGA (SEQ ID NO:81);
CCGCGCCCATGCCG (SEQ ID NO:82);
GCGCCCATGCCGA (SEQ ID NO:83);
CCGCGCCCATGCC (SEQ ID NO:84);
CGCCCATGCCGA (SEQ ID NO:85);
CCGCGCCCATGC (SEQ ID NO:86);
GCCCATGCCGA (SEQ ID NO:87);
CCGCGCCCATG (SEQ ID NO:88);
CCCATGCCGA (SEQ ID NO:89);
CCGCGCCCAT (SEQ ID NO:90);
CCATGCCGA (SEQ ID NO:91); and
CCGCGCCCA (SEQ ID NO:92).
Given the benefit of the present disclosure, the skilled artisan would also be able now, in similar fashion, to prepare any and all possible n-mers from each of the disclosed sequences, and from these sequences, identify and select those particular oligonucleotide sequences that specifically inhibit xcex21-AR-specific mRNA expression for therapeutic use by using an acceptable in vitro and/or in vivo assay, such as those described hereinbelow.
Likewise, based upon the sequence of the human xcex21-AR gene, the inventors have identified highly preferred sequences of from about 15 to about 25 nucleotides in length that specifically bind to the mRNA encoding xcex21-AR polypeptide, and that reduce the level of polypeptide in a cell expressing such an mRNA. Illustrative sequences in this size range are identified below:
Length=15 nucleotides:
5xe2x80x2-CACCCCCGCGCCCAT-3xe2x80x2 (SEQ ID NO:93)
5xe2x80x2-ACCCCCGCGCCCATG-3xe2x80x2 (SEQ ID NO:94)
5xe2x80x2-CGCGCCCATGCCGAG-3xe2x80x2 (SEQ ID NO:95)
5xe2x80x2-GCGCCCATGCCGAGC-3xe2x80x2 (SEQ ID NO:96)
5xe2x80x2-CGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:97)
5xe2x80x2-GCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:98)
5xe2x80x2-CCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:99)
5xe2x80x2-CCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:100)
5xe2x80x2-CATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:101)
5xe2x80x2-ATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:102)
Length=16 nucleotides:
5xe2x80x2-GCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:103)
5xe2x80x2-CGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:104)
5xe2x80x2-GCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:105)
5xe2x80x2-CCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:106)
5xe2x80x2-CCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:107)
5xe2x80x2-CATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:108)
5xe2x80x2-ATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:109)
5xe2x80x2-TGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:110)
Length=17 nucleotides:
5xe2x80x2-CGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:111)
5xe2x80x2-GCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:112)
5xe2x80x2-CGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:113)
5xe2x80x2-GCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:114)
5xe2x80x2-CCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:115)
5xe2x80x2-CCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:116)
5xe2x80x2-CATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:117)
5xe2x80x2-ATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:118)
Length=18 nucleotides:
5xe2x80x2-CCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:119)
5xe2x80x2-CGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:120)
5xe2x80x2-GCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:121)
5xe2x80x2-CGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:122)
5xe2x80x2-GCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:123)
5xe2x80x2-CCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:124)
5xe2x80x2-CCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:125)
5xe2x80x2-CATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:126)
Length=19 nucleotides:
5xe2x80x2-ACCCCCGCGCCCATGCCGA-3xe2x80x2 (SEQ ID NO:127)
5xe2x80x2-CCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:128)
5xe2x80x2-CCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:129)
5xe2x80x2-CGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:130)
5xe2x80x2-GCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:131)
5xe2x80x2-CGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:132)
5xe2x80x2-GCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:133)
5xe2x80x2-CCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:134)
5xe2x80x2-CCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:135)
Length=20 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGA-3xe2x80x2 (SEQ ID NO:136)
5xe2x80x2-ACCCCCGCGCCCATGCCGAG-3xe2x80x2 (SEQ ID NO:137)
5xe2x80x2-CCCCCGCGCCCATGCCGAGC-3xe2x80x2 (SEQ ID NO:138)
5xe2x80x2-CCCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:139)
5xe2x80x2-CCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:140)
5xe2x80x2-CCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:141)
5xe2x80x2-CGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:142)
5xe2x80x2-GCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:143)
5xe2x80x2-CGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:144)
5xe2x80x2-GCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:145)
5xe2x80x2-CCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:146)
Length=21 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGAG-3xe2x80x2 (SEQ ID NO:147)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGC-3xe2x80x2 (SEQ ID NO:148)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:149)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:150)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:151)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:152)
5xe2x80x2-CGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:153)
5xe2x80x2-GCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:154)
5xe2x80x2-CGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:155)
5xe2x80x2-GCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:156)
Length=22 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGAGC-3xe2x80x2 (SEQ ID NO:157)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:158)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:159)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:160)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:161)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:162)
5xe2x80x2-CGCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:163)
5xe2x80x2-GCGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:164)
5xe2x80x2-CGCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:165)
Length=23 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGAGCT-3xe2x80x2 (SEQ ID NO:166)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:167)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:168)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:169)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:170)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:171)
5xe2x80x2-CGCGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:172)
5xe2x80x2-GCGCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:173)
Length=24 nucleotides:
5xe2x80x2-CACCCCCGCGCCCATGCCGAGCTG-3xe2x80x2 (SEQ ID NO:174)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:175)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:176)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:177)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:178)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:179)
5xe2x80x2-CGCGCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:180)
Length=25 nucleotides:
5xe2x80x2-CACCCCCCGCGCCATGCCGAGCTGC-3xe2x80x2 (SEQ ID NO:181)
5xe2x80x2-ACCCCCGCGCCCATGCCGAGCTGCG-3xe2x80x2 (SEQ ID NO:182)
5xe2x80x2-CCCCCGCGCCCATGCCGAGCTGCGG-3xe2x80x2 (SEQ ID NO:183)
5xe2x80x2-CCCCGCGCCCATGCCGAGCTGCGGA-3xe2x80x2 (SEQ ID NO:184)
5xe2x80x2-CCCGCGCCCATGCCGAGCTGCGGAG-3xe2x80x2 (SEQ ID NO:185)
5xe2x80x2-CCGCGCCCATGCCGAGCTGCGGAGG-3xe2x80x2 (SEQ ID NO:186)